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United States Patent |
6,232,299
|
Jirousek
,   et al.
|
May 15, 2001
|
Use of protein kinase C inhibitors to enhance the clinical efficacy of
oncolytic agents and radiation therapy
Abstract
A method for treating neoplasms is disclosed, particularly using the
.beta.-isozyme selective PKC inhibitor, (S)-3,4-[N,
N'-1,1'-((2"-ethoxy)-3'"-(O)-4'"-(N,N-dimethylamino)-butane)-bis-(3,3'-ind
olyl)]-1(H)-pyrrole-2,5-dione or one of its salts, such PKC inhibitors
enhance the clinical efficacy of oncolytic agents and radiation therapy.
Inventors:
|
Jirousek; Michael R. (Indianapolis, IN);
Stramm; Lawrence E. (Indianapolis, IN);
Ways; Douglas Kirk (Indianapolis, IN)
|
Assignee:
|
Eli Lilly and Company (Indianapolis, IN)
|
Appl. No.:
|
841738 |
Filed:
|
April 30, 1997 |
Current U.S. Class: |
514/49; 424/649; 514/183; 514/185 |
Intern'l Class: |
A61K 031/70; A61K 031/33; A61K 031/555; A61K 033/24 |
Field of Search: |
514/183,185,49
424/649
|
References Cited
U.S. Patent Documents
4937232 | Jun., 1990 | Bell et al. | 514/26.
|
4990519 | Feb., 1991 | Jones et al. | 514/510.
|
5057614 | Oct., 1991 | Davis et al. | 514/510.
|
5141957 | Aug., 1992 | Jiang et al. | 514/510.
|
5204370 | Apr., 1993 | Jiang et al. | 514/475.
|
5216014 | Jun., 1993 | Jiang et al. | 514/455.
|
5270310 | Dec., 1993 | Bell et al. | 514/238.
|
5461146 | Oct., 1995 | Lewis et al. | 514/211.
|
5481003 | Jan., 1996 | Gillig et al. | 514/414.
|
5488167 | Jan., 1996 | Hudlicky | 546/489.
|
5491242 | Feb., 1996 | Gillig et al. | 548/455.
|
5545636 | Aug., 1996 | Heath, Jr. et al. | 514/214.
|
5552391 | Sep., 1996 | Coutts et al. | 514/44.
|
5552396 | Sep., 1996 | Heath, Jr. et al. | 514/183.
|
5578590 | Nov., 1996 | Grunicke et al. | 514/200.
|
5616577 | Apr., 1997 | Nambi et al. | 514/215.
|
5621098 | Apr., 1997 | Heath, Jr. et al. | 540/472.
|
5621101 | Apr., 1997 | Lewis et al. | 514/445.
|
Foreign Patent Documents |
0 657 411 A1 | Feb., 1994 | EP.
| |
Other References
Bundgaard, H. Design of Prodrugs, (1985).
Vente, et al., Cell Growth & Differentiation, (1995), 6: 371-382.
Ways, et al., Cell Growth & Differentiation, (1994), 5: 1195-1203.
Carter et al., Chemotherapy of Cancer, 2 nd edition, John Wiley & Sons, pp.
79-80, Aug. 3, 1981.
|
Primary Examiner: Goldberg; Jerome D.
Attorney, Agent or Firm: Darkes; Paul R., Caltrider; Steven P.
Parent Case Text
This application claims the priority benefits of the U.S. Provisional
application Serial No. 60/016,658 filed May 1, 1996.
Claims
What is claimed is:
1. A method for treating a neoplastic condition sensitive to the
combination below which comprises administering to a mammal in need of
such treatment an effective amount of an oncolytic agent having an
anti-neoplastic effect in combination with a protein kinase C inhibitor
selective for a beta-1 or beta-2 isozyme of protein kinase C, wherein the
protein kinase C inhibitor is administered in an amount sufficient to
enhance the anti-neoplastic effect of the oncolytic agent, and wherein the
protein kinase C inhibitor is a bis-indolylmaleimide or a macrocyclic
bis-indolylmaleimide and has the following formula:
##STR5##
wherein:
W is --O--, --S--, --SO--, --SO.sub.2 --, --CO--, C.sub.2 -C.sub.6
alkylene, substituted alkylene, C.sub.2 -C.sub.6 alkenylene, aryl-,
-aryl(CH.sub.2).sub.m O--, -heterocycle-, -heterocycle-(CH.sub.2).sub.m
O--, -fused bicyclic-, -fused bicyclic- (CH.sub.2).sub.m O--, --NR.sup.3
--, --NOR.sup.3 --, --CONH--, or --NHCO--;
X and Y are independently C.sub.1 -C.sub.4 alkylene, substituted alkylene,
or together X, Y, and W combine to form --(CH.sub.2).sub.n -AA-;
R's are hydrogen or up to four optional substituents independently selected
from halo, C.sub.1 -C.sub.4 alkyl, hydroxy, C.sub.1 -C.sub.4 alkoxy,
haloalkyl, nitro, NR.sup.4 R.sup.5, or --NHCO(C.sub.1 -C.sub.4 alkyl);
R.sup.2 is hydrogen, CH.sub.3 CO--, NH.sub.2, or hydroxy;
R.sup.3 is hydrogen, (CH.sub.2).sub.m aryl, C.sub.1 -C.sub.4 alkyl,
--COO(C.sub.1 -C.sub.4 alkyl), --CONR.sup.4 R.sup.5, --(C.dbd.NH)NH.sub.2,
--SO(C.sub.1 -C.sub.4 alkyl), --SO.sub.2 (NR.sup.4 R.sup.5), or --SO.sub.2
(C.sub.1 -C.sub.4 alkyl);
R.sup.4 and R.sup.5 are independently hydrogen, C.sub.1 -C.sub.4 alkyl,
phenyl, benzyl, or combine to the nitrogen to which they are bonded to
form a saturated or unsaturated 5 or 6 member ring;
AA is an amino acid residue;
m is independently 0, 1, 2, or 3; and
n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,
prodrug or ester thereof.
2. The method of claim 1 wherein the protein kinase C inhibitor has the
following formula:
##STR6##
wherein Z is --(CH.sub.2).sub.p -- or --(CH.sub.2).sub.p
--O--(CH.sub.2).sub.p --; R.sup.4 is hydroxy, --SH, C.sub.1 -C.sub.4
alkyl, (CH.sub.2).sub.m aryl, --NH(aryl), --N(CH.sub.3) (CF.sub.3),
--NH(CF.sub.3), or --NR.sup.5 R.sup.6 ; R.sup.5 is hydrogen or C.sub.1
-C.sub.4 alky; R.sup.6 is hydrogen, C.sub.1 -C.sub.4 alkyl or benzyl; p is
0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically
acceptable salt, prodrug or ester thereof.
3. The method of claim 1 wherein the protein kinase C inhibitor has the
following formula:
##STR7##
wherein Z is --(CH.sub.2).sub.p --; R.sup.4 is --NR.sup.5 R.sup.6,
--NH(CF.sub.3), or --N(CH.sub.3) (CF.sub.3); R.sup.5 and R.sup.6 are
independently H or C.sub.1 -C.sub.4 alkyl; p is 0, 1, or 2; and m is
independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or
ester thereof.
4. The method of claim 1, wherein the protein kinase C inhibitor comprises
(S)-3,4-[N,
N'-1,1'-((2"-ethoxy)-3'"(O)4'"-(N,N-dimethylamino)-butane)-bis-(3,3'-indol
yl)]-1(H)-pyrrole-2,5-dione or its pharmaceutically acceptable acid salt.
5. The method of claim 1, wherein the oncolytic agent is selected from the
group consisting of 1-.beta.-D-arabinofuranosylcytosine, etoposide,
cis-platinum, adriamycin, 2-chloro-2-deoxyadenosine,
9-.beta.-D-arabinosyl-2-fluoroadenine, and glucocorticoids.
6. A method for treating a neoplastic condition sensitive to the
combination below which comprises administering to a mammal in need of
such treatment an effective amount of 1-.beta.-D-arabinofuranosylcytosine
in combination with a protein kinase C inhibitor which comprises
(S)-3,4-[N,N'-
1,1'-((2"-ethoxy)-3'"(O)-4'"-(N,N-dimethylamino)-butane)-bis-(3,3'-indolyl
1)]-1(H)-pyrrole-2,5-dione or its pharmaceutically acceptable acid salt,
wherein the protein kinase C inhibitor is administered in an amount
sufficient to enhance the anti-neoplastic effect of the
1-.beta.-D-arabinofuranosylcytosine.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly directed to a method for enhancing
anti-neoplasm effects of chemotherapies and radiation therapies with PKC
inhibitors. The present invention is particularly directed to the use of
Protein Kinase C (PKC) inhibitors, especially a particular class of
isozyme selective PKC inhibitors in combination with an oncolytic agent or
.gamma.-irradiation to enhance their anti-neoplasm effects in treatment of
neoplasms.
2. Description of Related Art
Therapeutic treatments have been developed over the years to treat
neoplasms. There are two major approaches to treat neoplasms: 1)
chemotherapy employing oncolytic agents, and 2) radiation therapy, e.g.,
.gamma.-irradiation. Oncolytic agents and .gamma.-irradiation cause
cytotoxic effects, preferentially to tumor cells, and cause cell death.
Studies have shown that .gamma.-irradiation and certain groups of oncolytic
agents assert their cytotoxic effects by activating programmed cell death
or apoptosis. A balance between the activities of apoptotic and
antiapoptotic intracellular signal transduction pathways is important
towards a cell's decision of undergoing apoptosis in response to the above
mentioned chemotherapy as well as radiation therapy.
PKC inhibitors has been proposed for cancer therapy, e.g. see U.S. Pat. No.
5,552,391, and PKC activities have been indicated to exert antiapoptotic
effects, especially in response to radiation therapies, e.g.,
.gamma.-irradiation. In particular, studies have shown that activation of
PKC inhibits apoptosis induced by anti-neoplasm agents such as Ara-c,
2-chloro-2-deoxyadenosine, 9-.beta.-D-arabinosyl-2-fluoroadenine, and
.gamma.-irradiation therapy. There also have been indications that down
regulation of PKC activities in tumor cells enhances apoptosis stimulated
by oncolytic agents. PKC activation has been shown to attenuate
.gamma.-irradiation induced cell death.
There is a need in the art to develop therapeutic agents which enhance the
apoptotic signal transduction pathways in cells and thereby enhance the
clinical efficacy of oncolytic agents and radiation therapy.
SUMMARY OF INVENTION
It is an object of the invention to provide methods for treating a
neoplasm.
It is another object of the invention to provide methods for enhancing an
anti-neoplasm effect of an oncolytic agent.
It is still another object of the invention to provide methods for
enhancing anti-neoplasm effects of radiation therapy.
These and other objects of the invention are provided by one or more of the
embodiments described below.
In one embodiment of the invention there is provided a method for treating
a neoplasm which comprises administrating to a mammal in need of such
treatment an oncolytic agent or .gamma.-irradiation in combination with a
protein kinase C inhibitor.
In still another embodiment of the invention there is provided a method for
enhancing an anti-neoplasm effect of chemotherapy and radiation therapy
which comprises administrating a protein kinase C inhibitor in combination
with said oncolytic agent or radiation therapy.
The present invention provides the art with a method for increasing
apoptotic effects in cells and is thus effective in enhancing the
anti-neoplasm effects of chemotherapies and radiation therapies.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the dosage effect of bryostatin 1 on PKC isoforms.
FIG. 2 demonstrates the incubation time effect of bryostatin 1 on PKC
isoforms.
FIG. 3 demonstrates that down regulation of PKC-.beta. enhances the
efficacy of .gamma.-irradiation.
FIG. 4 shows that increased expression of PKC-.beta. demonstrates
resistance to radiation stimulated cell death.
DETAILED DESCRIPTION OF THE INVENTION
It is a discovery of the present invention that use of PKC inhibitors,
especially a particular class of protein kinase C inhibitors, reduces or
inhibits anti-apoptotic effects in a cell. Consequently, such compounds
can be used to enhance the anti-neoplasm effects of chemotherapies and
radiation therapies.
The method of this invention may employ any PKC inhibitor known in the art
including non-specific PKC inhibitors and specific PKC inhibitors of
different isoforms. Informations about PKC inhibitors, and methods for
their preparation are readily available in the art. For example, different
kinds of PKC inhibitors and their preparation are described in U.S. Pat.
Nos. 5,621,101, 5,621,098, 5,616,577, 5,578,590, 5,545,636, 5,491,242,
5,488,167, 5,481,003, 5,461,146, 5,270,310, 5,216,014, 5,204,370,
5,141,957, 4,990,519, and 4,937,232, all of which are incoporated herein
by reference. Preferably the present invention utilizes those protein
kinase C inhibitors that effectively inhibit the .beta. isozyme. One
suitable group of compounds are generally described in the prior art as
bis-indolylmaleimides or macrocyclic bis-indolylmaleimides.
Bis-indolymaleimides well recognized in the prior art inlcude those
compounds described in U.S. Pat. Nos. 5,621,098, 5,552,396, 5,545,636,
5,481,003, 5,491,242, and 5,057,614, all incorporated by reference herein.
Macrocyclic bis-indolylmaleimides are particularly represented by the
compounds of formula I. These compounds, and methods for their
preparation, have been disclosed in U.S. Pat. No. 5,552,396, which is
incorporated herein by reference. In accordance with the present
invention, these compounds are administered in combination with other
anti-neoplasm therapies to a mammal in need of such treatment. In
particular, these compounds can be used to enhance the anti-neoplasm
effects of chemotherapies and radiation therapies.
One preferred class of compounds for use in the method of the invention has
the formula:
##STR1##
wherein:
W is --O--, --S--, --SO--, --SO.sub.2 --, --CO--, C.sub.2 -C.sub.6
alkylene, substituted alkylene, C.sub.2 -C.sub.6 alkenylene, -aryl-,
-aryl(CH.sub.2).sub.m O--, -heterocycle-, -heterocycle-(CH.sub.2).sub.m
O--, -fused bicyclic-, -fused bicyclic-(CH.sub.2).sub.m O--, --NR.sup.3
--, --NOR.sup.3 --, --CONH--, or --NHCO--;
X and Y are independently C.sub.1 -C.sub.4 alkylene, substituted alkylene,
or together X, Y, and W combine to form --(CH.sub.2).sub.n --AA--;
R.sup.1 s are hydrogen or up to four optional substituents independently
selected from halo, C.sub.1 -C.sub.4 alkyl, hydroxy, C.sub.1 -C.sub.4
alkoxy, haloalkyl, nitro, NR.sup.4 R.sup.5, or --NHCO(C.sub.1 -C.sub.4
alkyl);
R.sup.2 is hydrogen, CH.sub.3 CO--, NH.sub.2, or hydroxy;
R.sup.3 is hydrogen, (CH.sub.2).sub.m aryl, C.sub.1 -C.sub.4 alkyl,
--COO(C.sub.1 -C.sub.4 alkyl), --CONR.sup.4 R.sup.5, --(C.dbd.NH)NH.sub.2,
--SO(C.sub.1 -C.sub.4 alkyl), --SO.sub.2 (NR.sup.4 R.sup.5), or --SO.sub.2
(C.sub.1 -C.sub.4 alkyl);
R.sup.4 and R.sup.5 are independently hydrogen, C.sub.1 -C.sub.4 alkyl,
phenyl, benzyl, or combine to the nitrogen to which they are bonded to
form a saturated or unsaturated 5 or 6 member ring;
AA is an amino acid residue;
m is independently 0, 1, 2, or 3; and
n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt,
prodrug or ester thereof.
A more preferred class of compounds for use in this invention is
represented by formula I wherein the moieties --X--W--Y-- contain 4 to 8
atoms, which may be substituted or unsubstituted. Most preferably, the
moieties --X--W--Y-- contain 6 atoms.
Other preferred compounds for use in the method of this invention are those
compounds of formula I wherein R.sup.1 and R.sup.2 are hydrogen; and W is
a substituted alkylene, --O--, S--, --CONH--, --NHCO-- or --NR.sup.3 --.
Particularly preferred compounds are compounds of the formula Ia:
##STR2##
wherein Z is --(CH.sub.2).sub.p -- or --(CH.sub.2).sub.p
--O--(CH.sub.2).sub.p --; R.sup.4 is hydroxy, --SH, C.sub.1 -C.sub.4
alkyl, (CH.sub.2).sub.m aryl, --NH(aryl), --N(CH.sub.3) (CF.sub.3),
--NH(CF.sub.3), or --NR.sup.5 R.sup.6 ; R.sup.5 is hydrogen or C.sub.1
-C.sub.4 alky; R.sup.6 is hydrogen, C.sub.1 -C.sub.4 alkyl or benzyl; p is
0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically
acceptable salt, prodrug or ester thereof. Most preferred compounds of the
formula Ia are those wherein Z is CH.sub.2 ; and R.sup.4 is --NH.sub.2,
--NH(CF.sub.3), or --N(CH.sub.3).sub.2.
Other preferred compounds for use in the method of the present invention
are compounds wherein W in formula I is --O--, Y is a substituted
alkylene, and X is an alkylene. These preferred compounds are represented
by formula Ib:
##STR3##
wherein Z is --(CH.sub.2).sub.p --; R.sup.4 is --NR.sup.5 R.sup.6,
--NH(CF.sub.3), or --N(CH.sub.3) (CF.sub.3); R.sup.5 and R.sup.6 are
independently H or C.sub.1 -C.sub.4 alkyl; p is 0, 1, or 2; and m is
independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or
ester thereof. Most preferred compounds of formula Ib are those wherein p
is 1; and R.sup.5 and R.sup.6 are methyl.
Because they contain a basic moiety, the compounds of formulae I, Ia, and
Ib can also exist as pharmaceutically acceptable acid addition salts.
Acids commonly employed to form such salts include inorganic acids such as
hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, as
well as organic acids such as para-toluenesulfonic, methanesulfonic,
oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic,
acetic acid, and related inorganic and organic acids. Such
pharmaceutically acceptable salts thus include sulfate, pyrosulfate,
bisulfate, sulfite, bisulfite, phosphate, mono-hydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,
iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,
isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate,
suberate, sebacate, fumarate, maleate, 2-butyne-1,4-dioate, 3-hexyne-2,
5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate,
phthalate, xylenesulfonate, phenylacetate, phenylpropionate,
phenylbutyrate, citrate, lactate, hippurate, .beta.-hydroxybutyrate,
glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like.
Particularly the hydrochloric and mesylate salts are used.
In addition to pharmaceutically-acceptable salts, other salts also can
exist. They may serve as intermediates in the purification of the
compounds, in the preparation of other salts, or in the identification and
characterization of the compounds or intermediates.
The pharmaceutically acceptable salts of compounds of formulae I, Ia, and
Ib can also exist as various solvates, such as with water, methanol,
ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such
solvates can also be prepared. The source of such solvate can be from the
solvent of crystallization, inherent in the solvent of preparation or
crystallization, or adventitious to such solvent.
It is recognized that various stereoisomeric forms of the compounds of
formulae I, Ia, and Ib may exist; for example, W may contain a chiral
carbon atom in the substituted alkylene moiety. The compounds are normally
prepared as racemates and can conveniently be used as such. Alternatively,
both individual enantiomers can be isolated or synthesized by conventional
techniques if so desired. Such racemates and individual enantiomers and
mixtures thereof form part of the compounds used in the methods of the
present invention.
The compounds utilized in this invention also encompass the
pharmaceutically acceptable prodrugs of the compounds of formulae I, Ia,
and lb. A prodrug is a drug which has been chemically modified and may be
biologically inactive at its site of action, but which may be degraded or
modified by one or more enzymatic or other in vivo processes to the parent
bioactive form. This prodrug likely may have a different pharmacokinetic
profile than the parent, enabling easier absorption across the mucosal
epithelium, better salt formation or solubility, and/or improved systemic
stability (an increase in plasma half-life, for example). Typically, such
chemical modifications include the following:
1) ester or amide derivatives which may be cleaved by esterases or lipases;
2) peptides which may be recognized by specific or nonspecific proteases;
or
3) derivatives that accumulate at a site of action through membrane
selection of a prodrug form or a modified prodrug form; or any combination
of 1 to 3, supra. Conventional procedures for the selection and
preparation of suitable prodrug derivatives are described, for example, in
H. Bundgaard, Design of Prodrugs, (1985).
The synthesis of various bis-indole-N-maleimide derivatives is described in
Davis et al. U.S. Pat. No. 5,057,614 and the synthesis of the preferred
compounds suitable for use in this invention are described in the
previously identified U.S. Pat. No. 5,552,396 and in Faul et al. EP
publication 0 657 411 A1, all of which are incorporated herein by
reference.
One particularly preferred protein kinase C inhibitor for use in the method
of this invention is the compound described in Example 5g
((S)-3,4-[N,N'-1,1'-((2"-ethoxy)-3'"(O)-4'"-(N,N-dimethylamino)-butane)-bi
s-(3,3'-indolyl)]-1(H)-pyrrole-2,5-dione Hydrochloride Salt) of the
aforementioned U.S. Pat. No. 5,552,396. This compound is a potent protein
kinase C inhibitor. It is selective to protein kinase C over other kinases
and is highly isozyme-selective, i.e., it is selective for the beta-1 and
beta -2 isozymes. Other salts of this compound also would be favored,
especially the mesylate salts.
A preferred mesylate salt can be prepared by reacting a compound of the
formula II
##STR4##
with methanesulfonic acid in a non-reactive organic solvent, preferably an
organic/water mixture, and most preferably water-acetone. Other solvents
such as methanol, acetone, ethylacetate and mixtures thereof are operable.
The ratio of solvent to water is not critical and generally determined by
the solubility of the reagents. Preferred solvent to water ratios are
generally from 0.1:1 to 100:1 solvent to water by volume. Preferably, the
ratio is 1:1 to 20:1 and most preferably 5:1 to 10:1. The optimal ratio is
dependent on the solvent selected and is preferably acetone at a 9:1
solvent to water ratio.
The reaction usually involves approximately equimolar amounts of the two
reagents, although other ratios, especially those wherein the
methanesulfonic acid is in excess, are operative. The rate of addition of
methanesulfonic acid is not critical to the reaction and may be added
rapidly (<5 minutes) or slowly over 6 or more hours. The reaction is
carried out at temperatures ranging from 0.degree. C. to reflux. The
reaction mixture is stirred until formation of the salt is complete, as
determined by x-ray powder diffraction and can take from 5 minutes to 12
hours.
The salts of the present invention are preferably and readily prepared as a
crystalline form. The trihydrate form of the salt may be readily converted
to the monohydrate upon drying or exposure to 20-60% relative humidity.
The salt is substantially crystalline demonstrating a defined melting
point, birefringence, and an x-ray diffraction pattern. Generally, the
crystals have less than 10% amorphous solid and preferably less than 5%
and most preferably less than 1% amorphous solid.
The mesylate salt is isolated by filtration or other separation techniques
appreciated in the art directly from the reaction mixture in yields
ranging from 50% to 100%. Recrystallization and other purification
techniques known in the art may be used to further purify the salt if
desired.
The PKC inhibitors, including the compounds described above, are used in
combination with conventional anti-neoplasm therapies to treat mammals,
especially humans with neoplasia. The procedures for conventional
anti-neoplasm therapies, including chemotherapies, e.g. using oncolytic
agents and radiation therapies e.g., .gamma.-irradiation are known,
readily available, and routinely practiced in the art, e.g., see
Harrison's PRINCIPLES OF INTERNAL MEDICINE 11th edition, McGraw-Hill Book
Company.
Neoplasia is characterized by abnormal growth of cells which often results
in the invasion of normal tissues, e.g., primary tumors or the spread to
distant organs, e.g., metastasis. The treatment of any neoplasia by
conventional anti-neoplasm therapies can be enhanced by the present
invention. Such neoplastic growth includes but not limited to primary
tumors, primary tumors that are incompletely removed by surgical
techniques, primary tumors which have been adequately treated but which
are at high risk to develop a metastatic disease subsequently, and an
established metastatic disease.
Specifically, the PKC inhibitors described above can enhance the
anti-neoplasm effects of an oncolytic agent. The wide variety of available
oncolytic agents are contemplated for combination therapy in accordance
with present invention. In a preferred embodiment, oncolytic agents that
assert their cytotoxic effects by activating programmed cell death or
apoptosis are used in combination with the described PKC inhibitors. These
include but not limited to 1-.beta.-D-arabinofuranosylcytosine or Ara-c,
etoposide or VP-16, cis-diamminedichloroplatinum (II) or cis-platinum,
doxorubicin or adriamycin, 2-chloro-2-deoxyadenosine,
9-.beta.-D-arabinosyl-2-fluoroadenine, and glucocorticoids.
All the neoplastic conditions treatable with such oncolytic agents can be
treated in accordance with the present invention by using a combination of
a PKC inhibitor with one or more oncolytic agents. The oncolytic agents
assert the cytotoxicity or anti-neoplasm effects in a variety of specific
neoplastic conditions. For example, Ara-c is normally used for treatment
of childhood-null acute lymphoid leukemia (ALL), thymic ALL, B-cell ALL,
acute myeloid leukemia, acute granulocytic leukemia and its variants,
non-Hodgkins lymphoma, myelomonocytoid leukemia, acute megakaryocytoid
leukemia and Burkitt's lymphoma, Adult-B-ALL, acute myeloid leukemia,
chronic lymphoid leukemia, chronic myeloid leukemia, and T cell leukemia.
VP-16 is normally used for treatment of testicular carcinoma, small and
large non-small cell lung carcinoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, choriocarcinoma, Ewing's sarcoma, and acute granulocytic
leukemia. Cis-platinum can be employed for treatment of testicular
carcinoma, germ cell tumors, ovarian carcinomas, prostate cancer, lung
cancer, sarcomas, cervical cancer, endometrial cancer, gastric cancer,
breast cancer, and cancer of the head and neck. 2-Chloro-2-deoxyadenosine
and 9-.beta.-D-arabinosyl-2-fluoroadenine can be used to treat chronic
lymphoid leukemia, lymphomas and hairy cell leukemia. Doxorubicin can be
used to treat acute granulocytic leukemia and its variants, ALL, breast
cancer, bladder cancer, ovarian cancer, thyroid cancer, lung cancer,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, sarcomas, gastric carcinoma,
prostate cancer, endometrial cancer, Wilm's tumor and neuroblastoma.
Clinical effects of oncolytic agents in all neoplastic conditions
treatable with oncolytic agents including the ones discussed above can be
potentiated by use of a combination therapy with the identified PKC
inhibitors in accordance with the present invention.
The PKC inhibitors identified in the present invention can also enhance the
anti-neoplasm effects of a radiation therapy. Usually .gamma.-irradiation
is used to treat the site of a solid tumor directly.
Experimental results provided in the present invention demonstrate that the
complete down regulation or loss of protein kinase C-.beta. is associated
with the synergistical enhancement of the oncolytic induced apoptosis in
human leukemic cells (FIG. 1). Similarly, significant down regulation of
protein kinase C-.beta. in U937 human leukemic cells enhances radiation
stimulated cell death (FIG. 2). U937 human leukemic cells that overexpress
protein kinase C-.beta. demonstrate resistance to radiation stimulated
cell death (FIG. 3). These data provide a strong indication that the PKC
inhibitors, especially .beta. isozyme selective inhibitors, preferably
used in accordance with the present invention can enhance tumor killing or
the anti-neoplasm effects of chemotherapies and radiation therapies and
improve clinical responses to these currently used therapeutic modalities.
The PKC inhibitors of the present invention are administered in combination
with other anti-neoplasm therapies including oncolytic agents and
radiation therapy. The phrase "in combination with other therapies" means
that the compounds can be administered shortly before, shortly after, or
concurrent with such other anti-neoplasm therapies. The compounds can be
administered in combination with more than one anti-neoplasm therapy. In a
preferred embodiment, the compounds are administered from 2 weeks to 1 day
before any chemotherapy, or 2 weeks to 1 day before any radiation therapy.
Alternatively, the PKC inhibitors can be administered during
chemotherapies and radiation therapies. If administered following
chemotherapy or radiation therapy, the PKC inhibitors should be given
within 1 to 14 days following the primary treatments.
One skilled in the art will recognize that the amount of PKC inhibitor to
be administered in accordance with the present invention in combination
with other anti-neoplasm agents or therapies is that amount sufficient to
enhance the anti-neoplasm effects of oncolytic agents or radiation
therapies or that amount sufficient to induce apoptosis or cell death.
Such amount may vary inter alia, depending upon the size and the type of
neoplasia, the concentration of the compound in the therapeutic
formulation, the specific anti-neoplasm agents used, the timing of the
administration of the PKC inhibitors relative to the other therapies, and
the age, size and condition of the patient.
Both in vivo and in vitro tests can be used to assess the amount of the
compounds needed for inducing apoptosis. For example, human leukemic cells
could be exposed in vitro to various concentrations of oncolytic agents,
e.g. Ara-c, or to radiation in the presence or absence of the PKC
inhibitor compounds used in the present invention. Appropriate neoplastic
cell types can be chosen for different oncolytic agents. Other protein
kinase C selective inhibitors can also be used for comparison. At various
time points, cells would be examined for viability by conventional methods
or by any means available in the art. Apoptosis or cell death can be
measured by any means known in the art. Cell death can be determined and
quantified via trypan blue exclusion, and reduced clonogenecity in soft
agar. As well understood by those skilled in the technology, apoptosis is
a specific mode of cell death recognized by a characteristic pattern of
morphological, biochemical, and molecular changes including but not
limited to, endonucleolysis (DNA ladder), abnormal DNA breaks, and
condensation of chromatin and cytoplasm (condensed and punctate nuclei).
These changes can be readily detected by any means known in the art, e.g.,
microscopy; flow cytometric methods based on increased sensitivity of DNA
to denaturation and altered light scattering properties; DNA fragmentation
as assessed by agarose gel electrophoresis; terminal DNA transferase
assay, (TdT assay), and nick translation assay (NT assay).
In vivo studies can be done using tumor xenografts inoculated into
immunocompromised or sygenic animals. After inoculation and growth of the
primary implant, the animals would be treated with the compounds in the
present invention prior to exposure to the desired oncolytic or radiation
treatment. The size of the tumor implant before and after each treatment
in the presence and absence of the compounds in the present invention can
be used as an indication of the therapeutic efficacy of the treatment.
Generally, an amount of protein kinase C inhibitor to be administered in
combination with other anti-neoplasm therapies is decided on a case by
case basis by the attending physician. As a guideline, the extent of the
neoplasia, the body weight, and the age of the patient will be considered,
among other factors, when setting an appropriate dose. Normally, the PKC
inhibitors of the present invention are expected to potentiate the
anti-neoplasm effects of oncolytic agents and radiation therapy from about
2 fold to about 10 fold.
Generally, a suitable dose is one that results in a concentration of the
protein kinase C inhibitor at the site of tumor cells in the range of 0.5
nM to 200 .mu.M, and more usually from 20 nM to 80 nM. It is expected that
serum concentrations of 40 nM to 150 nM should be sufficient in most
circumstances.
To obtain these treatment concentrations, a patient in need of treatment
likely will be administered between about 0.1 mg per day per kg of body
weight and 1.5 mg per day per kg. Usually, not more than about 1.0 mg per
day per kg of body weight of protein kinase C inhibitor should be needed.
As noted above, the above amounts may vary on a case-by-case basis.
The compounds of formula I and the preferred compounds of formula Ia and Ib
are preferably formulated prior to administration. Suitable pharmaceutical
formulations are prepared by known procedures using well known and readily
available ingredients. In making the compositions suitable for use in the
method of the present invention, the active ingredient will usually be
mixed with a carrier, or diluted by a carrier, or enclosed within a
carrier which may be in the form of a capsule, sachet, paper or other
container. When the carrier serves as a diluent, it may be a solid,
semisolid or liquid material which acts as a vehicle, excipient or medium
for the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium),
soft and hard gelatin capsules, suppositories, sterile injectable
solutions and sterile packaged powders for either oral or topical
application.
Some examples of suitable carriers, excipient, and diluents include
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphates, alginate, tragacanth, gelatin, calcium silicate,
microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup,
methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium
stearate and mineral oil. The formulations can additionally include
lubricating agents, wetting agents, emulsifying and suspending agents,
preserving agents, sweetening agents or flavoring agents. The compositions
of the invention may be formulated so as to provide quick, sustained or
delayed release of the active ingredient after administration to the
patient. The compositions are preferably formulated in a unit dosage form,
each dosage containing from about 0.05 mg to about 3 g, more usually about
64 mg of the active ingredient. However, it will be understood that the
therapeutic dosage administered will be determined by the physician in the
light of the relevant circumstances including the severity of the
condition to be treated, the choice of compound to be administered and the
chosen route of administration. Therefore, the above dosage ranges are not
intended to limit the scope of the invention in any way. The term "unit
dosage form" refers to physically discrete units suitable as unitary
dosages for human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable pharmaceutical
carrier.
In addition to the above formulations, most of which may be administered
orally, the compounds used in the method of the present invention also may
be administered topically. Topical formulations include ointments, creams
and gels.
Ointments generally are prepared using either (1) an oleaginous base, i.e.,
one consisting of fixed oils or hydrocarbons, such as white petrolatum or
mineral oil, or (2) an absorbent base, i.e., one consisting of an
anhydrous substance or substances which can absorb water, for example
anhydrous lanolin. Customarily, following formation of the base, whether
oleaginous or absorbent, the active ingredient (compound) is added to an
amount affording the desired concentration.
Creams are oil/water emulsions. They consist of an oil phase (internal
phase), comprising typically fixed oils, hydrocarbons, and the like, such
as waxes, petrolatum, mineral oil, and the like, and an aqueous phase
(continuous phase), comprising water and any water-soluble substances,
such as added salts. The two phases are stabilized by use of an
emulsifying agent, for example, a surface active agent, such as sodium
lauryl sulfate; hydrophilic colloids, such as acacia colloidal clays,
veegum, and the like. Upon formation of the emulsion, the active
ingredient (compound) customarily is added in an amount to achieve the
desired concentration.
Gels comprise a base selected from an oleaginous base, water, or an
emulsion-suspension base. To the base is added a gelling agent which forms
a matrix in the base, increasing its viscosity. Examples of gelling agents
are hydroxypropyl cellulose, acrylic acid polymers, and the like.
Customarily, the active ingredient (compounds) is added to the formulation
at the desired concentration at a point preceding addition of the gelling
agent.
The amount of compound incorporated into a topical formulation is not
critical; the concentration should be within a range sufficient to permit
ready application of the formulation to the affected tissue area in an
amount which will deliver the desired amount of compound to the desired
treatment site.
The customary amount of a topical formulation to be applied to an affected
tissue will depend upon an affected tissue size and concentration of
compound in the formulation. Generally, the formulation will be applied to
the effected tissue in an amount affording from about 1 to about 500 .mu.g
compound per cm.sup.2 of an affected tissue. Preferably, the applied
amount of compound will range from about 30 to about 300 .mu.g/cm.sup.2,
more preferably, from about 50 to about 200 .mu.g/cm.sup.2, and, most
preferably, from about 60 to about 100 .mu.g/cm.sup.2.
The following formulation examples are illustrative only and are not
intended to limit the scope of the invention in any way.
Formulation 1
Hard gelatin capsules are prepared using the following ingredients:
Quantity
(mg/capsule)
Active agent 250
starch, dried 200
magnesium stearate 10
Total 460 mg
The above ingredients are mixed and filled into hard gelatin capsules in
460 mg quantities.
Formulation 2
A tablet is prepared using the ingredients below:
Quantity
(mg/tablet)
Active agent 60 mg
starch 45 mg
microcrystalline cellulose 35 mg
polyvinylpyrrolidone 4 mg
(as 10% solution in water)
sodium carboxymethyl starch 4.5 mg
magnesium stearate 0.5 mg
talc 1 mg
Total 150 mg
The components are blended and compressed to form tablets each weighing 665
mg.
Formulation 3
Tablets each containing 60 mg of active ingredient are made as follows:
Quantity
(mg/capsule)
Active agent 250
cellulose, microcrystalline 400
silicon dioxide, fumed 10
stearic acid 5
Total 665 mg
The active ingredient, starch and cellulose are passed through a No. 45
mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone
is mixed with the resultant powders which are then passed through a No. 14
mesh U.S. sieve. The granules so produced are dried at 50.degree. C. and
passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch,
magnesium stearate and talc, previously passed through a No. 60 mesh U.S.
sieve, are then added to the granules which, after mixing, are compressed
on a tablet machine to yield tablets each weighing 150 mg.
EXAMPLES
Example 1
Effects of Bryostatin to PKC Isoforms
This experiment demonstrates the dosage and time effects of bryostatin to
PKC isoforms.
Human leukemia cells U937 in the amount of 0.5.times.10.sup.6 were treated
with various amount of bryostatin 1 for 24 hours. Subsequently, the cells
were solubilized for preparation of protein samples according to a routine
procedure. The protein samples from bryostatin treated cells were then
used in Western blot analysis with a protein kinase C-.beta. specific
antiserum previously described in Ways et al., Cell Growth &
Differentiation 1994, 5: 1195-1203. As shown in FIGS. 1 and 2, bryostatin
treatment caused PKC-.beta. activity to decrease within certain amount of
time, i.e., 10 nM bryostatin affects PKC-.beta. within 2 hours, or 1 nM
bryostatin affects PKC-.beta. within 24 hours. In a repeated experiment,
similar results were obtained.
Example 2
The Enhanced Efficacy of .gamma.-irradiation Caused by PKC-.beta. Down
Regulation
This experiment demonstrates that PKC-.beta. down regulation enhances the
efficacy of .gamma.-irradiation.
Human leukemia cells U937 were treated for 24 hours with either 3 nM
bryostatin 1 or the control solution, i.e., the vehicle for bryostatin 1.
The cells were then irradiated with either 500 or 1000 rads of
.gamma.-irradiation. Seventy-two hours after irradiation, cellular
viability was examined using propidium iodide exclusion and quantified by
FACS analysis as previously described in Ways et al., Cell Growth &
Differentiation 1994, 5: 1195-1203. Viability assays were performed in
triplicate. As shown in FIG. 3, .gamma.-irradiation-induced apoptosis was
enhanced under the condition when PKC-.beta. was significantly
down-regulated using bryostatin 1. Similar results were obtained in
several repeated experiments.
Example 4
Cells Overexpressing PKC-.beta. Demonstrate Resistance to Radiation
Stimulated Cell Death
Parental U937 cells and U937 PKC-.zeta. overexpressing cells (PKC-.zeta.
cells) were treated with 0, 500, or 1000 rads of .gamma.-irradiation. It
is known that PKC-.zeta. cells display increased level of PKC-.beta. (Ways
et al., Cell Growth & Differentiation, 1994, 5:1195-1203). Seventy two
hours after irradiation, cellular viability was examined using propidium
iodide exclusion and quantified by FACS analysis as previously described
in Ways et al., Cell Growth & Differentiation, 1995, 6: 371-382. Viability
assays were performed in triplicate. As shown in FIG. 4, cells having an
increased level of PKC-.beta. demonstrated resistance to radiation
stimulated cell death. Similar results were obtained in several repeated
experiments.
The principles, preferred embodiments and modes of operation of the present
invention have been described in the foregoing specification. The
invention which is intended to be protected herein, however, is not to be
construed as limited to the particular forms disclosed, since they are to
be regarded as illustrative rather than restrictive. Variations and
changes may be made by those skilled in the art without departing from the
spirit of the invention.
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